Scholarly article on topic 'Vascular biology of ageing—Implications in hypertension'

Vascular biology of ageing—Implications in hypertension Academic research paper on "Clinical medicine"

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{"Vascular remodeling" / "Endothelial dysfunction" / "Oxidative stress" / Mitochondria}

Abstract of research paper on Clinical medicine, author of scientific article — Adam Harvey, Augusto C. Montezano, Rhian M. Touyz

Abstract Ageing is associated with functional, structural and mechanical changes in arteries that closely resemble the vascular alterations in hypertension. Characteristic features of large and small arteries that occur with ageing and during the development of hypertension include endothelial dysfunction, vascular remodelling, inflammation, calcification and increased stiffness. Arterial changes in young hypertensive patients mimic those in old normotensive individuals. Hypertension accelerates and augments age-related vascular remodelling and dysfunction, and ageing may impact on the severity of vascular damage in hypertension, indicating close interactions between biological ageing and blood pressure elevation. Molecular and cellular mechanisms underlying vascular alterations in ageing and hypertension are common and include aberrant signal transduction, oxidative stress and activation of pro-inflammatory and pro-fibrotic transcription factors. Strategies to suppress age-associated vascular changes could ameliorate vascular damage associated with hypertension. An overview on the vascular biology of ageing and hypertension is presented and novel molecular mechanisms contributing to these processes are discussed. The complex interaction between biological ageing and blood pressure elevation on the vasculature is highlighted. This article is part of a Special Issue entitled: CV Ageing.

Academic research paper on topic "Vascular biology of ageing—Implications in hypertension"


YJMCC-08067; No. of pages: 10; 4C: 2,4, 6

Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

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Journal of Molecular and Cellular Cardiology

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Review article

Vascular biology of ageing—Implications in hypertension

Adam Harvey, Augusto C. Montezano, Rhian M. Touyz *

Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, UK



Article history:

Received 20 January 2015

Received in revised form 30 March 2015

Accepted 9 April 2015

Available online xxxx


Vascular remodeling Endothelial dysfunction Oxidative stress Mitochondria

Ageing is associated with functional, structural and mechanical changes in arteries that closely resemble the vascular alterations in hypertension. Characteristic features of large and small arteries that occur with ageing and during the development of hypertension include endothelial dysfunction, vascular remodelling, inflammation, calcification and increased stiffness. Arterial changes in young hypertensive patients mimic those in old normo-tensive individuals. Hypertension accelerates and augments age-related vascular remodelling and dysfunction, and ageing may impact on the severity of vascular damage in hypertension, indicating close interactions between biological ageing and blood pressure elevation. Molecular and cellular mechanisms underlying vascular alterations in ageing and hypertension are common and include aberrant signal transduction, oxidative stress and activation of pro-inflammatory and pro-fibrotic transcription factors. Strategies to suppress age-associated vascular changes could ameliorate vascular damage associated with hypertension. An overview on the vascular biology of ageing and hypertension is presented and novel molecular mechanisms contributing to these processes are discussed. The complex interaction between biological ageing and blood pressure elevation on the vasculature is highlighted. This article is part of a Special Issue entitled: CV Ageing.

© 2015 Published by Elsevier Ltd.


1. Introduction..............................................................................................................................0

2. Structural and mechanical changes in the ageing vasculature..................................................................................0

3. Vascular calcification......................................................................................................................0

4. Ageing associated vascular inflammation....................................................................................................0

5. Vascular contractility and ageing............................................................................................................0

6. Endothelial function and ageing............................................................................................................0

7. Vascular signalling in ageing..............................................................................................................0

7.1. Sirtuins............................................................................................................................0

7.2. PGC-1a............................................................................................................................0

7.3. FoxO transcription factors..........................................................................................................0

7.4. p66shc............................................................................................................................0

7.5. Cell cycle regulators, senescence and autophagy......................................................................................0

7.6. Mitogen-activated protein kinases (MAPK)............................................................................................0

7.7. Oxidative stress in vascular ageing..................................................................................................0

7.8. Endoplasmic reticulum stress in vascular ageing......................................................................................0

7.9. Vascular changes in hypertension recapitulate those in ageing..........................................................................0

7.10. Effects of pro-hypertensive stimuli on vascular ageing: the renin-angiotensin-aldosterone system (RAAS)............................0

7.11. Effects of novel anti-hypertensive factors on vascular ageing..........................................................................0

7.12. Vascular damage in hypertension may be independent of ageing......................................................................0

7.13. Lessons learned from children with hypertension....................................................................................0

8. Summary and conclusions................................................................................................................0

* Corresponding author at: Institute of Cardiovascular and Medical Sciences, BHF Glasgow Cardiovascular Research Centre, University of Glasgow, 126 University Place, Glasgow, G12 8TA, UK. Tel.: +44 141 330 7775/7774; fax: + 44141 330 3360. E-mail address: (R.M. Touyz). 0022-2828/© 2015 Published by Elsevier Ltd.


A. Harvey et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx




1. Introduction

Clinical studies show a significant relationship between ageing and increased blood pressure, with advancing age being a major non-modifiable risk factor in the development of hypertension [1]. This is due, in part, to changes that occur in the vasculature, including endothe-lial dysfunction, vascular remodelling, increased vascular stiffness and inflammation. These functional and structural changes define the 'vascular phenotype' of hypertension, features that are also found during ageing [2] (Fig. 1). At the cellular level, there is endothelial cell damage, increased vascular smooth muscle cell growth, cell migration, inflammation, contraction, extracellular matrix deposition, fibrosis, and calcification [3].

Young patients with elevated blood pressure exhibit arterial changes similar to those in older individuals with normal blood pressure, and accordingly the concept of 'premature' or 'early' vascular ageing in hypertension has been proposed [4]. Hypertension accelerates age-related vascular changes, processes that are attenuated when blood pressure is normalised. The direct relationship between ageing and vascular health is evident in progeria syndrome, where patients exhibit accelerated ageing, endothelial dysfunction, accelerated atherosclerosis and die prematurely from complications of cardiovascular disease, such as stroke and myocardial infarction [5]. Considering the fact that the population is ageing and that the major chronic disease of ageing is hypertension and associated cardiovascular complications, the potential health and economic burden in our modern society is enormous. Accordingly it is important to understand how vascular function changes with ageing and how this impacts on hypertension, so that targeted strategies could be developed to prevent and repair damaged 'aged' arteries and thereby reduce the risk of hypertension and target organ

Young - Healthy

f \ Normal Vascular Homeostasis

damage. In the present review, we discuss the vascular changes that occur with ageing and during the development of hypertension and focus on some molecular mechanisms that underlie these vascular changes.

2. Structural and mechanical changes in the ageing vasculature

Physiological changes to the vascular wall are dynamic and occur throughout life [6,7]. Endothelial cell turnover occurs over years, whereas that of vascular smooth muscle cells seems to occur over a shorter time period. Many structural and mechanical alterations have been observed in the aged vasculature including increased intimal-to-media (IM) thickness, evidenced by the finding that the IM thickness of the carotid artery increases two- to three-fold between 20 and 90 years of age [8,9]. Subclinical IM thickening is strongly associated with ageing and is also a predictor of future cardiovascular events [8,9]. Both aortic length and circumference gradually increase with advancing age [10-12]. Associated with these structural alterations are mechanical changes, characterised by a reduction in compliance, reduced elasticity/ distensibility and increased stiffness [8,9]. Stiffening of the large conduit arteries due to fracture of elastin fibres within the tunica media and collagenous remodelling, results in increased aortic pulse pressure and pulse wave velocity (PWV). Increased PWV, a non-invasive measure of vascular stiffness, increases in both sexes with ageing and is determined by the mean arterial pressure and the intrinsic stress/strain relationship (stiffness) of the arterial wall. As arterial wall stiffness increases, central systolic pressure increases and diastolic pressure decreases, leading to increased pulse pressure, an independent risk factor for future cardiovascular events [13]. Processes underlying these structural and mechanical changes involve growth and migration of vascular smooth muscle cells

Aged - Hypertension

r Endothelial dysfunction^ ^ M:L ratio Vascular remodelling Increased stiffness Vascular inflammation _Calcification J

Fig. 1. Schematic demonstrating vascular changes that occur during ageing and with the development of hypertension. Vascular changes in hypertension mimic those found in arteries observed with ageing.


A. Harvey et al./ Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

within the media, vascular calcification and changes in the ratio of collagen and elastin in the vascular wall. Physiologically, the ratio of collagen and elastin remains constant due to their gradual production and degradation. In aged rodents the absolute elastin content of the aorta was shown not to differ to young counterparts. However, increased collagen content in 30-month-old animals compared to 6-month-old animals meant that the relative elastin content was decreased [14]. Collagen and elastin are regulated by catabolic matrix metalloproteinases (MMPs). Throughout ageing the balance between MMPs and their inhibitors (TIMPs) changes. For example, increased MMP-2 expression and activity in the vessels of old rats and non-human primates is increased compared to young counterparts [15,16].

3. Vascular calcification

Vascular calcification is a tightly controlled process similar to bone formation, where mineralization of the internal elastic lamina and elastic fibres in the media results in vascular stiffening. Calcification of the vascular media is a hallmark of vascular ageing [17]. Upregulation of transcription factors such as cbfa1 (core-binding factor 1a)/Runx2, MSX-2 and bone morphogenetic protein 2 (BMP-2), are involved in normal bone development and vascular calcification by regulating the expression of osteogenic proteins, including osteocalcin, osteonectin, alkaline phosphatase, collagen-1, and bone sialoprotein [18,19]. Another mechanism contributing to vascular mineralization is loss of calcification inhibitors, such as fetuin-A, matrix Gla protein, pyrophosphate, and osteopontin [19-21]. Molecular processes underlying this remain to be fully defined but increased expression of BMP2 and the osteoblast transcription factor Runx2/Cbfa1 [22], and modulation of Ca2+ and Mg2+ transport through cation channels, such as TRPM7 may represent important mediators in this process [23,24]. A correlation between age and vascular calcification has been described from 5% in individuals younger than 50 years to > 12% in individuals older than 80 years [25]. Ageing-associated vascular calcification has been reported in the aorta of rodents where the associated mechanisms include dysregulation of Matrix Gla protein [26]. Further possible mechanisms contributing to increased calcification with ageing includes dysregulation of vascular pyrophosphate [27,28]. Human studies have shown a weak inverse correlation between age and plasma pyrophosphate [29,30].

4. Ageing associated vascular inflammation

With ageing there is a shift towards a proinflammatory vascular phenotype with upregulation of inflammatory cytokines, chemokines and adhesion molecules in the vascular wall [8,9,31-38]. Pro-inflammatory transcription factors and proteins that have been identified in the ageing vascular media include MCP-1, TGF-(31, MMP-2, AP-1 and NF-kB [8,9,39]. Expression and activation of these molecules increases with ageing, processes that are usually associated with increased generation of reactive oxygen species (ROS). In aged arteries, there is downregulation of the transcription factor, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), which stimulates expression of antioxidant enzymes, thereby leading to decreased anti-oxidant potential and increased ROS bioavailability with consequent oxidative stress [40]. Oxidative stress is a potent inducer of redox-sensitive pro-inflammatory signalling pathways, further contributing to inflammation and vascular damage with ageing [36-40].

5. Vascular contractility and ageing

Functionally, vascular contraction is altered during ageing and is determined in large part by changes in vascular smooth muscle cell cytoskeletal organisation and impaired contractile signalling. Mesenteric arteries from aged rats demonstrate hypercontractility in response to phenylephrine compared to young controls [40] an effect which is mirrored in the aorta [41]. These findings are paralleled in studies utilising

the senescence-accelerated mouse (SAM-P8), which demonstrate increased vascular contractility in response to phenylephrine [42]. Conversely, studies performed on carotid vessels from aged guinea pigs displayed reduced contractile response to both phenylephrine and endothelin-1 (ET-1) compared to younger controls [43]. Thus it appears that differential responses during ageing may differ between species.

At the cellular level, with ageing, vascular smooth muscle cells, which are normally contractile, undergo phenotypic changes to become stiff and pro-migratory. Subsets of apoptotic, senescent and proliferative cells as well as hyper-contractile cells may co-exist in the vascular media. A major trigger for these functional changes is an increase in intracellular free Ca2+ concentration ([Ca2+]i), which occurs following activation of phopholipase C (PLC) leading to the generation of second messengers insitol trisphosphate (IP3) and diacylglycerol (DAG) [39,44]. Ca2+ binds to calmodulin facilitating an interaction with myosin light chain kinase (MLCK) leading to its activation. Activated MLCK triggers phosphorylation of the regulatory light chains of myosin (MLC20) promoting cycling of myosin cross-bridges with actin and consequent contraction. Dephosphorylation of MLC20 by myosin light chain phosphatase (MLCP) results in VSMC relaxation. As such, the relative activities of MLCK and MLCP determine vascular smooth muscle tone by influencing the degree of MLC20 phosphorylation. Arteries from aged animals display altered responses to various contractile agents including norepinephrine, serotonin and KCL [44,45]. Mechanisms for this are incompletely understood, but the percentage of phosphorylated MLC20 induced by vasoactive agonists is different in young versus aged rats and may play a role in altered age-related contractile responses [45].

6. Endothelial function and ageing

The vascular endothelium is a monolayer of cells that lines blood vessels and plays a key role in arterial function through synthesis and release of biologically active molecules that can influence endothelial function in an autocrine or paracrine fashion. The healthy endothelium is characterised by a vasodilatory, anti-inflammatory and antithrombotic phenotype. Endothelial dysfunction is characterised by reduced vasodilatory responses to flow or agonists and is proinflammatory. Independent of the occurrence of other pathologies, ageing results in altered endothelium-dependent relaxation of both the aorta and resistance arteries in rodents [46,47]. These findings have been corroborated in human studies that suggest that endothelial function is gradually compromised with ageing [48,49]. A primary mechanism responsible for the deterioration of endothelial function with ageing is thought to be reduced bioavailability of the endothelium derived relaxing factor, nitric oxide (NO), due to its interaction with ROS to form peroxynitrite. Peroxynitrite oxidises BH4, an essential cofactor for NO synthesis by endothelial nitric oxide synthase (eNOS), to its inactive form resulting in reduced NO production. Furthermore, reduced BH4 can result in eNOS uncoupling whereby superoxide is produced in preference to NO. Reductions in BH4 levels have been reported in aged rodents [50].

7. Vascular signalling in ageing

Molecular mechanisms and cell signalling events underlying the structural and functional alterations observed during ageing are similar to those that occur in hypertension (Fig. 2). Many age/longevity-related molecules and signalling cascades have been described, of which a few of the novel systems are highlighted below.

7.1. Sirtuins

Sirtuins (SIRTs) are a family of NAD-dependent protein deacetylases and ribosyl transferases consisting of 7 members which are localised in the cytoplasm (SIRT1 and SIRT2), nucleus (SIRT1, SIRT2, SIRT 6 and SIRT 7) or mitochondria (SIRT3, SIRT4 and SIRT 5). SIRTs have been


A. Harvey et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

Pro-hypertensive Factors (RAS, ET-1, Aldosterone) Ï



Cell cycle regulators


Ca2+ Mg2+

Mitochondria dysfunction Noxactivation



Inflammatory Genes MAPK

N / /\ I \ / \ I

Senescence Apoptosis Autophagy

Anoikis Proliferation


iVasorelaxation î Vasoreactivity


Vascular calcification

Endothelial dysfunction

VCAM-1, ICAM-1 Cytokines Chemokines

Vascular inflammation


Fig. 2. Molecular and cellular mechanisms associated with vascular changes in ageing and hypertension. Activation of pro-fibrotic, pro-inflammatory, redox-sensitive and growth/apoptotic signalling pathways lead to changes in vascular structure, mechanics and function with resultant arterial remodelling, calcification, inflammation, stiffness and impaired vasoreactivity. These vascular alterations are common features during ageing and in hypertension. VCAM-1, vascular cell adhesion molecule-1; ICAM-1, intercellular adhesion molecule-1; MMP, matrix metallo-proteinases; TIMP, tissue inhibitor of metalloproteinase; RAS, renin angiotensin system; ET-1, endothelin-1 ; NO, nitric oxide.

238 implicated in various cellular processes associated with ageing, includ-

239 ing, apoptosis, inflammation and mitochondrial biogenesis. SIRTs are

240 able to modulate the ageing process in a number of species [51-53].

241 This is highlighted by the following: 1) SIRT1 protects against

242 phosphate-induced arterial calcification, possibly due to the inhibition

243 of osteoblastic transdifferentiation [54]; 2) mitochondrial localised

244 SIRT3 regulates many proteins that are important in the regulation of

245 mitochondrial function including pyruvate dehydrogenase, SOD2 and

246 cyclophilin D; 3) SIRT3 —/— mice exhibit accelerated cardiovascular

247 ageing [55] and 4) SIRT3 has vasoprotective effects through interaction

248 with FOXO3, which enhances mitochondrial antioxidant defence

249 systems [56].

250 7.2. PGC-la

251 Another emerging candidate implicated in age-related signalling in

252 the vasculature, is peroxisome proliferator-activated receptor gamma

253 coactivator-1a (PGC-1a), which plays an important role in regulating

254 mitochondrial biogenesis and turnover [57]. Because mitochondria re-

255 quire continuous recycling and regeneration throughout the lifespan

256 and are subject to continuous damage over time, regulation of mito-

257 chondrial biogenesis and turnover is critical for maintained energy

258 production and prevention of oxidative damage, and the promotion of

259 healthy ageing. Impaired mitochondrial biogenesis is an important in-

260 ducer of age-related changes in the endothelium and vascular smooth

261 muscle [58-60]. The aged vasculature displays reduced levels of PGC-

262 1a with consequent mitochondrial dysregulation of the electron trans-

263 port chain and other mitochondrial proteins leading to oxidative stress

264 and vascular injury [60]. Decreased AMPK activity may contribute to

reduced PGC-1a activation and impaired mitochondrial function associ- 265 ated with ageing [61]. 266

7.3. FoxO transcription factors 267

The FoxO family of Forkhead transcription factors are involved in 268 tumour suppression, energy metabolism, and longevity. Mammals ex- 269 press four FoxO isoforms, FoxO1, FoxO3, FoxO4 and FoxO6. FoxO1, 270 FoxO3 and FoxO4 are phosphorylated in an Akt-dependent manner 271 that promotes FoxO export from the nucleus to the cytoplasm, thereby 272 repressing FoxO transcriptional function. FoxO targets include genes 273 that have pivotal roles in cell cycle progression (p21, p27) and ROS de- 274 toxification (MnSOD) and thus may be important in regulation of the 275 ageing phenotype in the vasculature [62,63]. FoxO3 is a direct target 276 of SIRT3 deacetylation protecting mitochondria against age-related ox- 277 idative stress and promoting upregulation of genes that are essential 278 for mitochondrial homeostasis [64]. Several reports have suggested 279 that FoxO3 may be a determinant of ageing, due to the fact that 280 single-nucleotide polymorphisms in the FoxO3 gene are associated 281 with longevity in humans [65,66]. FoxO3 knockout mice however do 282 not exhibit reduced lifespan [67], and as such, the exact role of FoxO3 283 in longevity and ageing still remains unclear. 284

7.4. p66shc 285

Mitochondrial dysfunction and increased mitochondrial-derived 286 ROS have been implicated in vascular changes in ageing [68]. An impor- 287 tant mediator of mitochondrial ROS production and thus regulator of 288 the intracellular pathways that govern oxidative stress, apoptosis, and 289


A. Harvey etal./ Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

290 cell growth/survival is the adapter protein p66shc. p66Shc is phosphor-

291 ylated at serine 36 by PKQ3 and VEGF, resulting in recognition by the

292 prolyl isomerase Pin1, allowing translocation and entrance into mito-

293 chondria where it interacts with cytochrome C resulting in production

294 of H2O2. Levels of p66shc in heart, kidney and vascular smooth muscle

295 increase with ageing [69]. Mice lacking p66shc gene display a 30%

296 increase in lifespan compared to wild-type controls due to prevention

297 of oxidative stress and improved endothelial function [70,71 ].

298 7.5. Cell cycle regulators, senescence and autophagy

299 In culture, vascular cells respond to prolonged series of replication

300 and stresses by eventually entering an irreversible growth arrest or

301 senescent state [72]. After the Hayflick limit, cells enter an irreversible

302 cell cycle arrest in the G1 phase of the cell cycle and no longer respond

303 to growth stimuli. This phenomenon is called replicative senescence

304 and occurs in vascular ageing [73]. Senescent cells have a distinct

305 phenotype—they are large and flattened, express specific markers

306 ((-galactosidase), overexpress cell cycle molecular markers (p16 and

307 p21), form heterochromatic foci (yH2AX) and accumulate lipofuscin, a

308 non-degradable fluorescent compound [74]. Whilst the molecular mech-

309 anisms underlying cellular senescence have been the focus of numerous

310 studies, the impact of senescence in vivo has yet to be fully established,

311 especially since some studies show increased rates of vascular cell prolif-

312 eration in ageing and longevity [75,76].

313 Considering the remarkable plasticity of vascular smooth muscle

314 cells, there is a requirement for tight control of transcriptional, metabol-

315 ic and ultrastructural processes, events that are coordinated through

316 autophagy. Autophagy is the basic cellular mechanism that involves

317 cell degradation of unnecessary or dysfunctional molecules through

318 lysosomes [77]. In the vasculature, changes in autophagy have been

319 observed in experimental ageing [78].

320 7.6. Mitogen-activated protein kinases (MAPK)

321 Protein kinases are major regulators of signal transduction that

322 catalyse the phosphorylation of other proteins, thus regulating their ac-

323 tivity. Primary targets of protein kinases include transcription factors

324 which modulate intracellular signalling via specific alteration of down-

325 stream gene expression/activity [79]. A key group of protein kinases in

326 the vasculature are the serine/threonine sub-family, which act by pro-

327 moting phosphorylation of the OH group of serine or threonine residues

328 on target proteins [80]. Mitogen activated protein kinases (MAPKs)

329 represent a large family of proteins important in signal transduction

330 within the cardiovascular system, where they are involved in regulation

331 of a number of biological processes, such as cell migration, survival, ap-

332 optosis, proliferation, contraction and differentiation. MAPK signalling is

333 promoted by many stimuli including GPCR activation, receptor tyrosine

334 kinases, oxidative stress and growth factors, and comprises a number of

335 sequentially acting kinases which ultimately result in phosphorylation

336 and activation of terminal effector kinases, thereby transducing specific

337 cellular actions [80,81 ]. Several MAPK family subgroups have been iden-

338 tified, of which the major mammalian types appear to be ERK1/2, c-Jun

339 NH2-terminal kinases (JNK1, 2 and 3) and p38MAPK (a, (3, 8 and y),

340 which play key roles during cardiovascular development and vascular

341 function [82,83]. Several studies have demonstrated an age-dependent

342 increase in MAPK activation in vascular tissue [84,85].

343 77. Oxidative stress in vascular ageing

344 Common to many of the molecular and cellular processes described

345 above that underlie changes in the vasculature with ageing is oxidative

346 stress [86]. The concept that ROS are linked to ageing was suggested in

347 1 956 by Harman when he proposed the Free Radical Theory of Ageing,

348 stating that the accumulation of free radicals during ageing causes the

349 damage of biomolecules by these ROS and the development of

pathological disorders promoting cell senescence and organism ageing 350

[87,88]. Such processes are evident in vessels associated with ageing 351

and with hypertension [87,88]. Excessive production of ROS and reac- 352

tive nitrogen species (RNS) leads to oxidative modification of proteins, 353

DNA and lipids, which accumulate in cells leading to impaired cellular 354

and vascular function. In addition increased vascular ROS levels, togeth- 355 er with decreased eNOS-generated NO, compromise the vasodilatory 356

actions of NO and promote the formation of injurious peroxynitrite, 357

processes observed in aorta of aged rodents [89]. Oxidative stress is 358

critically involved in many of the molecular events of vascular ageing, 359

including: (1) increased pro-inflammatory responses in vascular cells, 360

(2) vascular dysfunction through oxidative modification of structural 361

and functional proteins regulating vascular contraction/relaxation, 362

fibrosis and calcification, (3) altered calcium homeostasis in vascular 363

cells, 4) activation of redox-sensitive pro-inflammatory and pro- 364

fibrotic transcription factors, and (4) activation of molecular mecha- 365 nisms leading to senescence and autophagy in endothelial and vascular 366

smooth muscle cells (Fig. 3). The fact that SOD mimetics, such as tempol, 367

normalise endothelial dysfunction in old rodents supports a role for in- 368 creased superoxide anion levels in age-related endothelial impairment 369

[90]. 370

Changes in cellular anti-oxidant systems are also important. The ex- 371

pression and activity of antioxidant enzymes, including SOD, decline as 372 tissues age. Decreased anti-oxidant capacity is further promoted by 373

downregulation of Nrf2, the master transcription factor regulating 374 anti-oxidant genes [91]. These processes are accompanied by chronic 375

low-grade inflammation mediated by redox-sensitive NFkB, which is 376

upregulated in aged vessels [92]. 377

Multiple oxidases generate ROS in the vascular wall and endotheli- 378

um, including NADPH oxidases (Nox), xanthine oxidase, uncoupled 379 NOS and mitochondrial oxidases. Of these, mitochondria seem to play 380

a major role in processes related to ageing. Noxs, of which there are 7 381

isoforms (Nox1-5, Duox1, Duox2), have also been shown to contribute 382

to oxidative stress in vascular ageing [93-95]. In particular, in aged 383

spontaneously hypertensive rat aortas, expression of Nox1 and Nox2, 384

but not of Nox4, was increased. This Nox upregulation was associated 385 with endothelial dysfunction, which was reversed by VAS2870, a Nox 386

inhibitor [96]. Noxs appear to be more important in pathological vascu- 387

lar remodelling associated with hypertension and cardiovascular 388

diseases [97-99]. Vascular xanthine oxidase and cytochrome P45 0 389 epoxygenases seem to be less important, since expression and activity 390

of these systems is not altered with ageing in humans [100]. 391

With biological ageing, mitochondria become dysfunctional leading 392

to reduced energy production and increased ROS formation. Mecha- 393

nisms related to mitochondrial dysfunction during ageing include 394

decreased ATP synthesis, increased apoptosis and mutations of mito- 395

chondrial DNA by oxidation [101]. During ageing, the electron flow in 396

mitochondria decreases, altering the oxygen consumption and inducing 397

ROS generation [101]. The pro-oxidative environment increases mito- 398 chondrial DNA damage, leading to further dysfunction of the respiratory 399

chain and more ROS production. Consequently, the rate ofapoptosis in- 400 creases, releasing an excessive amount of ROS into the cytosol, further 401

contributing to oxidative stress and vascular cell damage. 402

7.8. Endoplasmic reticulum stress in vascular ageing 403

Prolonged perturbation of the endoplasmic reticulum (ER) leads to 404 ER stress and unfolded protein response (UPR) and contributes to path- 405 ogenic processes associated with vascular damage and endothelial dys- 406

function [102]. The ER is an important site where proteins are folded 407 and post-translation modifications occur. It is also a site for Ca2+ storage 408

and cholesterol/lipid biosynthesis. Due to the large amount of unfolded 409

protein in the ER, a control system that avoids protein aggregation and 410

accumulation ofunfolded proteins is necessary. In experimental models 411 of ageing, the expression and activity of ER chaperones or folding 412

enzymes decay, whilst oxidative damage, such as carbonylation, is 413


6 A. Harvey et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

Vascular aging^Hypertension

Fig. 3. Role of reactive oxygen species (ROS) in vascular processes associated with ageing and hypertension. Pro-hypertensive factors, such as angiotensin II and endothelin-1, and biological ageing, increase ROS production in vascular cells. An increase in the levels of ROS lead to oxidation of proteins and DNA, affecting cell signalling and inducing injurious responses, such as inflammation, senescence, fibrosis, calcification, and hypertrophy in the vasculature. Oxidation of transcription factors that regulate the anti-oxidant capacity in vascular cells, such as Nrf2, are also affected by oxidation leading to decreased activity. Sources responsible for the increase in ROS generation and oxidative modification of cellular molecules are the mitochondria, NADPH oxidases (Nox) and endoplasmic reticulum (ER) stress.

exacerbated, leading to accumulation of misfolded/unfolded proteins and ER stress. This activates signalling mechanisms that are part of the UPR Induction of ER stress leads to endothelial cell apoptosis, but not senescence, implicated in endothelial dysfunction in ageing [103,104]. Inhibition of ER stress has been suggested as a novel therapeutic strategy to ameliorate vascular dysfunction during ageing [105]. However such approaches still require further investigation.

7.9. Vascular changes in hypertension recapitulate those in ageing

Many of the signalling pathways associated with vascular changes during ageing are also activated in hypertension leading to endothelial dysfunction, vascular inflammation, remodelling and increased arterial stiffness. With normal physiological ageing the process is gradual and regulated but in itself represents a strong and independent risk factor for hypertension and future cardiovascular events [106]. In susceptible individuals, due to genetic, environmental or in-utero factors 9fetal programming), processes underlying vascular changes are accelerated leading to 'early vascular ageing', which predisposes to cardiovascular disease. Numerous risk factors amplify the process of arterial ageing, including atherosclerosis, smoking, increased sodium intake and hypertension, due, in part, to increased oxidative stress, activation of proinflammatory and pro-fibrotic signalling pathways and upregulation of the renin-angiotensin-aldosterone system. As with ageing, experimental and human hypertension show a reduction in endothelium-dependent vasodilation, decreased NO bioavailability, NO synthase uncoupling, increased oxidative stress, telomere shortening and associated endothelial dysfunction. In arteries from aged humans, non-human

primates and rodents, expression of the AT[R is increased and sensitiv- 440

ity of the mineralocorticoid receptor to aldosterone is enhanced, 441

phenomena that are also observed in hypertension [107-109]. Ang II 442

promotes vascular calcification, inflammation, cell proliferation and 443

fibrosis and mimics age-associated vascular remodelling in young ro- 444

dents [110-112]. In large arteries these molecular and cellular processes 445

manifest as increased arterial stiffness, which is a major contributor to 446

elevated central blood pressure leading to isolated systolic hyperten- 447

sion, common in the elderly. Exactly what triggers these cellular and 448

vascular events remains unclear, and it is difficult to dissect out the 'age- 449

ing effect' from the 'blood pressure effect'. This 'conundrum of arterial 450

stiffness, elevated blood pressure and ageing' has recently been 451

reviewed by AlGhatrif and Lakatta [113], who concluded that vascular 452

properties depend on the net effect of multiple factors that are interde- 453

pendent and which change with ageing over a lifetime. 454

7.10. Effects of pro-hypertensive stimuli on vascular ageing: the renin- 455

angiotensin-aldosterone system (RAAS) 456

The RAAS plays an important role in functional, structural and 457

mechanical changes of the vasculature that occur with ageing and hy- 458

pertension [114]. This occurs through increased signalling via the AT 459

receptor. Expression of various components of the RAS, including 460

angiotensinogen, chymase, angiotensin converting enzyme (ACE) and 461

the AT1 receptor is increased in arteries of aged rodents and humans 462

[8,9]. To further support a role for the RAAS in the ageing process and 463

during hypertension are studies showing that ACE inhibitors and AT re- 464

ceptor blockers decrease ageing-associated vascular damage. Mice 465


A. Harvey et al./ Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

treated with enalapril or losartan demonstrated vasoprotection and an increase in life span [115,116]. Processes associated with these effects involve upregulation of NOS activity and increased NO production. An increase in antioxidant defences, such as SOD and glutathione, is another mechanism involved in the anti-ageing effects of the inhibition of the RAAS system, leading to an increase in NO bioavailability [117]. Moreover, lifelong treatment of young stroke-prone spontaneously hypertensive rats, with AT1 receptor blockers doubles the lifespan by improving endothelial function and alleviating complications of hypertension [118]. To further support a role for Ang II/AT1 receptor in oxida-tive stress, vascular injury and ageing, studies in mice with targeted disruption of the Agtr1a gene, which encodes the AT1A receptor, resulted in prolonged life span [119]. Agtr1a —/— mice developed less cardiac and vascular injury and oxidative damage was reduced compared with wild-type counterparts. The longevity phenotype was associated with an increased number of mitochondria and upregulation of pro-survival genes.

Clinical and experimental studies demonstrate that many pro-hypertensive systems influence processes of vascular ageing, including aldosterone, ET-1 and growth factors [120-124]. Arteries from aged rodents demonstrate upregulation of these systems, leading to stimulation of signalling pathways, oxidative stress, and activation of pro-inflammatory transcription factors, which promote a shift of endothelial and vascular smooth muscle cells to an ageing phenotype. On the other hand, infusion of Ang II, aldosterone or ET-1 in young animals, recapitulates arterial changes observed in aged animals.

7.11. Effects of novel anti-hypertensive factors on vascular ageing

NO is a potent vasodilator produced by endothelial cells that mediates vascular relaxation and thus plays a critical role in the regulation of blood pressure. Abnormalities in endothelial production of NO occur in hypertension and are due, in large part, to decreased eNOS activity [125]. NO donors such as glyceryl trinitrate (GTN) have been shown to possess anti-hypertensive properties [126] and evidence is emerging that NO and NO donors could confer beneficial effects on the phenotypic alterations that occur in the vasculature with ageing. For example, NO prevents differentiation of VSMCs into osteoblastic cells by inhibiting TGF-p [127]. The NO donor S-nitroso-penicillamine significantly reduces endothelial cell senescence and age-dependent inhibition of telomerase activity [128,129].

The gaseous messenger hydrogen sulphide (H2S) produced by cystathionine g-lyase (CSE) or cystathionine b-synthase (CBS) has recently emerged as a novel antihypertensive factor based on the observations that exogenous H2S is vasoprotective in pulmonary hypertension [130] and that it reduces systemic blood pressure by improving endothelial function [131]. CSE-deficient mice, have increased blood pressure and impaired endothelial function [132]. Mouse embryonic fi-broblasts from CSE knockout mice display accelerated cellular senescence and increased expression of p53 and p21, processes which were prevented by NaHS treatment. NaHS also enhanced Nrf2 nuclear translocation, and stimulated mRNA expression of Nrf2-targeted downstream anti-oxidant genes in this system, highlighting an important interplay between cellular ageing, senescence and oxidative stress [133]. Attenuation of endothelial cell senescence by H2S occurs through modulation of SIRT1 activity [134].

Plasma levels of H2S in humans decline with age [135] and several studies have shown that H2S protects against free radical-induced damage and exerts beneficial effects on age-associated diseases [136]. Several lines of evidence indicate that these beneficial effects may extend to vascular ageing, characterised by positive effects on many of the phenotypic vascular alterations that occur with advancing age. For example, the production of H2S is decreased in a rodent model of vascular calcification with the addition of H2S ameliorating this phenotype [137]. Further, in vascular smooth muscle cells, H2S was found to inhibit

calcium deposition in the extracellular matrix and suppress induction of osteoblastic transformation genes [138].

In endothelial cells stimulated with TNF-a, NaHS (H2S donor) suppressed pro-inflammatory responses by reducing the TNF-induced increase in expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin and E-selectin. Furthermore, TNF-a-induced NF-kB was decreased in the presence of NaHS [139]. H2S donors (NaHS and Na2S) can inhibit leukocyte adherence in mesenteric venules whilst inhibition of endogenous H2S synthesis promotes leukocyte adhesion and vascular inflammation [140].

7.12. Vascular damage in hypertension may be independent of ageing

Although there are many signalling pathways and functional and structural characteristics that are common in vessels during ageing and hypertension, these processes are dynamic and change throughout life and as such may not necessarily be superimposable. For example, with advancing age arterial stiffness and blood pressure start to diverge rather than parallel [141]. Also, circulating markers of inflammation including sVCAM-1, IL-6 and MCP-1 increase with age but do not necessarily correlate with elevations in blood pressure [142]. Furthermore, with ageing, aortic calcification is independently predictive of subsequent vascular morbidity and mortality beyond established risk factors with no evident correlation between calcification and systolic BP [143].

There is also some evidence to show that structural alterations in the vascular wall occur before the development of hypertension. For example rates of pulse wave velocity (PWV) increase are accelerated with both advancing age and elevated blood pressure. However, the effect of blood pressure on PWV increase occurs primarily during the prehypertensive phase and suggests that these vascular alterations precede the phase of established hypertension [8,144].

7.13. Lessons learned from children with hypertension

Vascular changes that occur with ageing may be independent of biological ageing in hypertension. This is highlighted in studies that have examined vascular function and arterial structure in children with hypertension. Endothelial dysfunction, arterial stiffening and structural alterations of the arterial wall may precede evidence of high blood pressure, as quantified by systolic and diastolic blood pressure, and may be independent of the ageing process [145]. This is evidenced by the findings that vascular injury is already present in children with mild hypertension, processes that are exaggerated as hypertension becomes more severe [146,147]. Functional alterations, including reduced endothelium-dependent vasorelaxation and decreased elasticity, seem to precede vascular structural changes. In obese pre-pubertal children, impaired brachial endothelial and vascular smooth muscle function is present without concomitant increase in carotid intima-to-media thickness. Functionally these changes lead to decreased vascular distensibil-ity and increased rigidity or stiffness. Arterial stiffness, as assessed by measurement of PWV, is increased in children with type 1 diabetes and in children with hypertension. Increased arterial stiffness in childhood hypertension is an important risk factor for severe hypertension and cardiovascular complications later in life. Results from the Amsterdam Growth and Health Longitudinal Study indicate that individuals with stiffer carotid arteries at 36 years of age were characterised during adolescence by increased blood pressure and increased PWV [148]. Factors that have been implicated in vascular dysfunction in childhood hypertension include activation of the sympathetic nervous system, adipokines, upregulation of the RAAS and increased oxidative stress, processes that also underlie physiological vascular ageing and EVA in adult hypertension [149-152].

Taken together, emerging experimental and clinical evidence indicates that although the molecular and cellular processes that characterise the vascular phenotype in hypertension resemble those that occur with normal healthy ageing, age per se may not be a critical


A. Harvey et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

591 factor. However, ageing may be a compounding factor that amplifies

592 vascular injury that occurs with blood pressure elevation. In the

593 presence of other co-morbidities, such as diabetes, dyslipidemia,

594 smoking and obesity, these processes may be further exaggerated.

595 8. Summary and conclusions

596 Ageing is associated with a progressive deterioration in endothelial

597 function, vascular remodelling, inflammation and increased arterial

598 stiffness. Processes underlying these processes include activation of

599 pro-inflammatory transcription factors, oxidative stress, cell senescence

600 and apoptosis, aberrant signalling cascades and a shift from a vasocon-

601 strictor to a proliferative vascular cell phenotype. Many of these

602 phenomena are also relevant in the pathophysiology of hypertension,

603 which is characterised by a vascular phenotype of impaired

604 endothelium-dependent vasorelaxation, arterial remodelling, increased

605 stiffness and vascular inflammation. Through such vascular changes,

606 ageing and hypertension are closely interlinked: ageing promotes

607 hypertension and pro-hypertensive factors promote vascular ageing.

608 Whilst many of the molecular processes and signalling pathways

609 contributing to vascular dysfunction are common in ageing and in hy-

610 pertension, biological age per se, may not be a fundamental factor,

611 since vascular damage is already present in children and young adults

612 with hypertension. A better understanding of the vascular biology of

613 ageing will facilitate development of strategies to promote healthy

614 vessels and suppress age-associated changes, especially in pathological

615 conditions. Such approaches could prevent or ameliorate vascular

616 damage in hypertension and hence reduce cardiovascular diseases,

617 commonly linked to ageing.

618 Disclosures

619 There are no disclosures to declare. Q4 Acknowledgements

621 Work from the author's laboratory was supported by grants 44018

622 and 57886, from the Canadian Institutes of Health Research (CIHR)

623 and grants from the British Heart Foundation (BHF). RMT is supported

624 through a BHF Chair. ACM is supported through a Leadership Fellowship

625 from the University of Glasgow.

626 References

627 [1] Wang M, Monticone RE, Lakatta EG. Arterial aging: a journey into subclinical

628 arterial disease. Curr Opin Nephrol Hypertens 2010;19(2):201-7.

629 [2] Barja G. Updating the mitochondrial free radical theory of aging: an integrated

630 view, key aspects, and confounding concepts. Antioxid Redox Signal 2013;

631 19(12):1420-45.

632 [3] Bachschmid MM, Schildknecht S, Matsui R, Zee R, Haeussler D, Cohen RA, et al.

633 Vascular aging: chronic oxidative stress and impairment of redox signalling—

634 consequences for vascular homeostasis and disease. Ann Med 2013;45(1):

635 17-36.

636 [4] Kotsis V, Stabouli S, Karafillis I, Nilsson P. Early vascular aging and the role of central

637 blood pressure. J Hypertens 2011;29(10):1847-53.

638 [5] Gerhard-Herman M, Smoot LB, Wake N, Kieran MW, Kleinman ME, Miller DT, et al.

639 Mechanisms of premature vascular aging in children with Hutchinson-Gilford

640 progeria syndrome. Hypertension 2012;59(1):92-7.

641 [6] Collins JA, Munoz JV, Patel TR Loukas M, Tubbs RS. The anatomy of the aging aorta.

642 Clin Anat 2014;27(3):463-6.

643 [7] Donato AJ, Gano LB, Eskurza I, Silver AE, Gates PE, Jablonski K, et al. Vascular endo-

644 thelial dysfunction with aging: endothelin-1 and endothelial nitric oxide synthase.

645 Am J Physiol Heart Circ Physiol 2009;297:H425-32.

646 [8] Lakatta EG, Levy D. Arterial and cardiac aging: major shareholders in cardiovascular

647 disease enterprises: Part I: aging arteries: a "setup" for vascular disease. Circulation

648 2003;107(1):139-46.

649 [9] Lakatta EG. The reality of aging viewed from the arterial wall. Artery Res 2013;7(2):

650 73-80.

651 [10] Gerstenblith G, FrederiksenJ, Yin FC, Fortuin NJ, Lakatta EG, Weisfeldt ML Echocar-

652 diographic assessment of a normal adult aging population. Circulation 1977;56(2):

653 273-8.

[11] Virmani R, Avolio AP, Mergner WJ, Robinowitz M, Herderick EE, Cornhill JF, et al. 654 Effect of aging on aortic morphology in populations with high and low prevalence 655 of hypertension and atherosclerosis. Comparison between occidental and Chinese 656 communities. AmJ Pathol 1991;139(5):1119-29. 657

[12] Sugawara J, Hayashi K, Yokoi T, Tanaka H. Age-associated elongation of the ascending 658 aorta in adults. JACC Cardiovasc Imaging 2008;1(6):739-48. 659

[13] Nilsson PM, Khalili P, Franklin SS. Blood pressure and pulse wave velocity as metrics 660 for evaluating pathologic ageing of the cardiovascular system. Blood Press 2014; 661 23(1):17-30. 662

[14] Michel J, Heudes D, Michel O, Poitevin P, Philippe M, Scalbert E, et al. Effect of 663 chronic ANG I-converting enzyme inhibition on aging processes. II. Large arteries. 664 AmJ Physiol 1994;267:R124-R124. 665

[15] Wang M, Takagi G, Asai K, Resuello RG, Natividad FF, Vatner DE, et al. Aging increases Q5 aortic MMP-2 activity and angiotensin II in nonhuman primates. Hypertension 2003 ; 667 41(6):1308-16. 668

[16] Li Z, Froehlich J, Galis ZS, Lakatta EG. Increased expression of matrix 669 metalloproteinase-2 in the thickened intima of aged rats. Hypertension 1999; 670 33(1):116-23. 671

[17] Jiang L, Zhang J, Monticone RE, Telljohann R, WuJ, WangM,etal.Calpain-1 regulation 672 of matrix metalloproteinase 2 activity in vascular smooth muscle cells facilitates age- 673 associated aortic wall calcification and fibrosis. Hypertension 2012;60(5):1192-9. 674

[18] Shanahan CM. Mechanisms of vascular calcification in CKD—evidence for prema- 675 ture ageing? Nat Rev Nephrol 2013;9(11):661-70. 676

[19] Alam MU, Kirton JP, Wilkinson FL, Towers E, Sinha S, Rouhi M, et al. Calcification is 677 associated with loss of functional calcium-sensing receptor in vascular smooth 678 muscle cells. Cardiovasc Res 2009;81 (2):260-8. 679

[20] Rosito GA, Massaro JM, Hoffmann U, Ruberg FL, Mahabadi AA, Vasan RS, et al. Peri- 680 cardial fat, visceral abdominal fat, cardiovascular disease risk factors, and vascular 681 calcification in a community-based sample: the Framingham Heart Study. Circulation 682 2008;117(5):605-13. 683

[21] McCarty MF, DiNicolantonio JJ. The molecular biology and pathophysiology of 684 vascular calcification. Postgrad Med 2014;126(2):54-64. 685

[22] Trebak M, Ginnan R, Singer HA, Jourd'heuil D. Interplay between calcium and reac- 686 tive oxygen/nitrogen species: an essential paradigm for vascular smooth muscle 687 signaling. Antioxid Redox Signal 2010;12(5):657-74. 688

[23] Louvet L, Büchel J, Steppan S, Passlick-Deetjen J, Massy ZA Magnesium prevents 689 phosphate-induced calcification in human aortic vascular smooth muscle cells. 690 Nephrol Dial Transplant 2013;28(4):869-78. 691

[24] Montezano AC, Zimmerman D, Yusuf H, Burger D, Chignalia AZ, Wadhera V, et al. 692 Vascular smooth muscle cell differentiation to an osteogenic phenotype involves 693 TRPM7 modulation by magnesium. Hypertension 2010 Sep;56(3):453-62. 694

[25] McClelland RL, Chung H, Detrano R, Post W, Kronmal RA. Distribution of coronary 695 artery calcium by race, gender, and age: results from the Multi-Ethnic Study of 696 Atherosclerosis (MESA). Circulation 2006;113(1):30-7. 697

[26] Sweatt A, Sane D, Hutson S, Wallin R Matrix Gla protein (MGP) and bone morpho- 698 genetic protein-2 in aortic calcified lesions of aging rats. J Thromb Haemost 2003; 699 1(1):178-85. 700

[27] Rutsch F, Nitschke Y, Terkeltaub R. Genetics in arterial calcification: pieces of a 701 puzzle and cogs in a wheel. Circ Res 2011;109(5):578-92. 702

[28] Leopold JA. Vascular calcification: an age-old problem of old age. Circulation 2013; 703 127(24):2380-2. 704

[29] Lomashvili KA, Khawandi W, O'Neill WC. Reduced plasma pyrophosphate levels in 705 hemodialysis patients. J Am Soc Nephrol 2005;16(8):2495-500. 706

[30] O'Neill wC, Sigrist MK, McIntyre CW. Plasma pyrophosphate and vascular calcification 707 in chronic kidney disease. Nephrol Dial Transplant 2010;25(1):187-91. 708

[31] Wang M, Monticone RE, Lakatta EG. Proinflammation of aging central arteries: a 709 mini-review. Gerontology 2014;60(6):519-29. 710

[32] Csiszar A, Ungvari Z, Edwards JG, Kaminski P, Wolin MS, Koller A, et al. Aging 711 induced phenotypic changes and oxidative stress impair coronary arteriolar function. 712 Circ Res 2002;90(11):1159-66. 713

[33] Bruunsgaard H, Skinh0j P, Pedersen AN, Schroll M, Pedersen B. Ageing, tumour 714 necrosis factor-alpha (TNF-a) and atherosclerosis. Clin Exp Immunol 2000; 715 121(2):255-60. 716

[34] Csiszar A, Ungvari Z. Synergistic effects of vascular IL-17 and TNFa may promote 717 coronary artery disease. Med Hypotheses 2004;63(4):696-8. 718

[35] Csiszar A, Labinskyy N, Smith K, Rivera A, Orosz Z, Ungvari Z. Vasculoprotective 719 effects of anti-tumor necrosis factor-a treatment in aging. Am J Pathol 2007; 720 170(1):388-98. 721

[36] Arenas IA, Xu Y, Davidge ST. Age-associated impairment in vasorelaxation to fluid 722 shear stress in the female vasculature is improved by TNF-alpha antagonism. Am 723 J Physiol Heart Circ Physiol 2006;290(3):H1259-63. 724

[37] Ungvari Z, Orosz Z, Labinskyy N, Rivera A, Xiangmin Z, Smith K, et al. Increased 725 mitochondrial H2O2 production promotes endothelial NF-kappaB activation in 726 aged rat arteries. AmJ Physiol Heart Circ Physiol 2007;293(1 ):H37-47. 727

[38] Paneni F, Costantino S, Cosentino F. Molecular pathways of arterial aging. Clin Sci 728 (Lond) 2015;128(2):69-79. 729

[39] Zicha J, Behuliak M, Pintérovâ M, Bencze M, Kunes J, Vanëckovâ I. The interaction of 730 calcium entry and calcium sensitization in the control of vascular tone and blood 731 pressure of normotensive and hypertensive rats. Physiol Res 2014;63:S19-27. 732

[40] Davidge ST, Hubel CA, McLaughlin MK. Impairment of vascular function is associat- 733 ed with an age-related increase of lipid peroxidation in rats. Am J Physiol Regul 734 Integr Comp Physiol 1996;271(6):R1625-31. 735

[41] Reyes-Toso CF, Obaya-Naredo D, Ricci CR, Planells FM, Pinto JE, Linares LM, et al. 736 Exp Gerontol 2007;42(4):337-42. 737

[42] Lloréns S, de Mera Raquel M, Melero-Fernandez, Pascual A, Prieto-Martin A, Q6 Mendizâbal Y, et al. The senescence-accelerated mouse (SAM-P8) as a model for 739


A. Harvey etal./ Journal of Molecular and

the study of vascular functional alterations during aging. Biogerontology 2007; 8(6):663-72.

de Andrade CR, Correa FM, de Oliveira AM. Aging and total stenosis triggers differential responses of carotid and basilar arteries to endothelin-1 and phenylephrine. J Smooth Muscle Res 2009;45(6):307-21.

Marchand A, Abi-Gerges A, Saliba Y, Merlet E, Lompre A. Calcium signaling in vascular smooth muscle cells: from physiology to pathology. Adv Exp Med Biol 2012;740:795-810.

Kitamura-Sasaka F, Ueda K, Kawai Y. Effects of aging on contraction and Ca2 mobilization in smooth muscle cells of the rat coronary artery. Yonago Acta Med 2001; 44(1):61-8.

Celermajer DS, Sorensen KE, Spiegelhalter DJ, Georgakopoulos D, Robinson J, Deanfield JE. Aging is associated with endothelial dysfunction in healthy men years before the age-related decline in women. J Am Coll Cardiol 1994;24(2): 471-6.

Dohi Y, Kojima M, Sato K, Lüscher TF. Age-related changes in vascular smooth

muscle and endothelium. Drugs Aging 1995;7(4):278-91.

Favero G, Paganelli C, Buffoli B, Rodella LF, Rezzani R. Endothelium and its

alterations in cardiovascular diseases: life style intervention. Biomed Res Int


Taddei S, Virdis A, Mattei P, Ghiadoni L, Gennari A, Fasolo CB, et al. Aging and endo-thelial function in normotensive subjects and patients with essential hypertension. Circulation 1995;91(7):1981-7.

Sindler AL, Delp MD, Reyes R Wu G, Muller-Delp JM. Effects of ageing an exercise training on eNOS uncoupling in skeletal muscle resistance arterioles. J Physiol 2009;587(Pt 15):3885-97.

Mostoslavsky R, Chua KF, Lombard DB, Pang WW, Fischer MR, Gellon L, et al. Genomic instability and aging-like phenotype in the absence of mammalian SIRT6. Cell 2006; 124(2):315-29.

Vakhrusheva O, Braeuer D, Liu Z, Braun T, Bober E. Sirt7-dependent inhibition of cell growth and proliferation might be instrumental to mediate tissue integrity during aging. J Physiol Pharmacol 2008;59(Suppl. 9):201-12. Zu Y, Liu L, Lee MY, Xu C, Liang Y, Man RY, et al. SIRT1 promotes proliferation and prevents senescence through targeting LKB1 in primary porcine aortic endothelial cells. Circ Res 2010;106(8):1384-93.

Takemura A, Iijima K, Ota H, Son BK, Ito Y, Ogawa S, et al. Sirtuin 1 retards hyperphosphatemia-induced calcification of vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2011;31(9):2054-62.

Hafner AV, Dai J, Gomes AP, Xiao CY, Palmeira CM, Rosenzweig A, et al. Regulation of the mPTP by SIRT3-mediated deacetylation of CypD at lysine 166 suppresses age-related cardiac hypertrophy. Aging (Albany NY) 2010;2(12):914-23. Tseng P, Hou S, Chen R, Peng H, Hsieh C, Kuo M, et al. Resveratrol promotes osteogenesis of human mesenchymal stem cells by upregulating RUNX2 gene expression via the SIRT1/FOXO3A axis. J Bone Miner Res 2011;26(10): 2552-63.

Mukherjee S, Basar MA, Davis C, Duttaroy A. Emerging functional similarities and divergences between Drosophila Spargel/dPGC-1 and mammalian PGC-1 protein. Front Genet 2014;5:216.

Mohamed SA, Hanke T, Erasmi AW, Bechtel MJ, Scharfschwerdt M, Meissner C, et al. Mitochondrial DNA deletions and the aging heart. Exp Gerontol 2006; 41(5):508-17.

Ungvari Z, Parrado-Fernandez C, Csiszar A, de Cabo R. Mechanisms underlying caloric restriction and lifespan regulation: implications for vascular aging. Circ Res 2008;102(5):519-28.

Zinovkin RA, Romaschenko VP, Galkin II, Zakharova VV, Pletjushkina OY, Chernyak BV, et al. Role of mitochondrial reactive oxygen species in age-related inflammatory activation of endothelium. Aging (Albany NY) 2014;6(8):661-74. Reznick RM, Zong H, Li J, Morino K, Moore IK, Yu HJ, et al. Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis. Cell Metab 2007;5(2):151-6.

van den Berg Maaike CW, Burgering BM. Integrating opposing signals toward Forkhead box O. Antioxid Redox Signal 2011 ;14(4):607-21. Oellerich MF, Potente M. FOXOs and sirtuins in vascular growth, maintenance, and aging. Circ Res 2012;110(9):1238-51.

Tseng AH, Shieh S, Wang DL. SIRT3 deacetylates FOXO3 to protect mitochondria

against oxidative damage. Free Radic Biol Med 2013;63:222-34.

Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, et al. FOXO3A genotype is

strongly associated with human longevity. Proc Natl Acad Sci U S A 2008;


Anselmi CV, Malovini A, Roncarati R, Novelli V, Villa F, Condorelli G, et al. Association of the FOXO3A locus with extreme longevity in a southern Italian centenarian study. Rejuvenation Res 2009;12(2):95-104.

Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science 2003; 301(5630):215-8.

Remmen HV, Richardson A. Oxidative damage to mitochondria and aging. Exp Gerontol 2001;36(7):957-68.

Lebiedzinska M, Duszynski J, Rizzuto R, Pinton P, Wieckowski M. Age-related changes in levels of p66Shc and serine 36-phosphorylated p66Shc in organs and mouse tissues. Arch Biochem Biophys 2009;486(1):73-80. Migliaccio E, Giorgio M, Mele S, Pelicci G, Reboldi P, Pandolfi PP, et al. The p66shc adaptor protein controls oxidative stress response and life span in mammals. Nature 1999;402(6759):309-13.

Francia P, delli Gatti C, Bachschmid M, Martin-Padura I, Savoia C, Migliaccio E, et al. Deletion of p66shc gene protects against age-related endothelial dysfunction. Circulation 2004;110(18):2889-95.

ellular Cardiology xxx (2015) xxx-xxx 9

[72] Minamino T, Yoshida T, Tateno K, Miyauchi H, Zou Y, Toko H, et al. Ras induces vascular smooth muscle cell senescence and inflammation in human atherosclerosis. Circulation 2003;108(18):2264-9.

[73] Campisi J, Robert L. Cell senescence: role in aging and age-related diseases. Interdiscip Top Gerontol 2014;39:45-61.

[74] Lauri A, Pompilio G, Capogrossi MC. The mitochondrial genome in aging and senescence. Ageing Res Rev 2014;18C:1-15.

[75] Rivard A, Principe N, Andres V. Age-dependent increase in c-fos activity and cyclin A expression in vascular smooth muscle cells. A potential link between aging, smooth muscle cell proliferation and atherosclerosis. Cardiovasc Res 2000;45(4): 1026-34.

[76] Vazquez-Padron RI, Lasko D, Li S, Louis L, Pestana IA, Pang M, et al. Aging exacerbates neointimal formation, and increases proliferation and reduces susceptibility to apo-ptosis of vascular smooth muscle cells in mice. J Vasc Surg 2004;40(6):1199-207.

[77] Salabei JK, Hill BG. Autophagic regulation of smooth muscle cell biology. Redox Biol 2014;4C:97-103.

[78] Gao L, Qi HB, Kc K, Zhang XM, Zhang H, Baker PN. Excessive autophagy induces the failure of trophoblast invasion and vasculature: possible relevance to the pathogenesis of preeclampsia. J Hypertens 2015;33(1):106-17.

[79] Force T, Hajjar R, Del Monte F, Rosenzweig A, Choukroun G. Signaling pathways mediating the response to hypertrophic stress in the heart. Gene Expr 1999;7(4-6):337.

[80] Cheng H, Force T. Why do kinase inhibitors cause cardiotoxicity and what can be done about it? Prog Cardiovasc Dis 2010;53(2):114-20.

[81] Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 1997;9(2):180-6.

[82] Bueno OF, De Windt LJ, Tymitz KM, Witt SA, Kimball TR, Klevitsky R, et al. The MEK1-ERK1/2 signaling pathway promotes compensated cardiac hypertrophy in transgenic mice. EMBO J 2000;19(23):6341-50.

[83] Zhang S, Weinheimer C, Courtois M, Kovacs A, Zhang CE, Cheng AM, et al. The role of the Grb2-p38 MAPK signaling pathway in cardiac hypertrophy and fibrosis. J Clin Invest 2003;111(6):833-42.

[84] Rice KM, Kinnard R, Harris R, Wright G, Blough E. Effects of aging on pressure-induced MAPK activation in the rat aorta. Pflugers Arch 2005;450(3):192-9.

[85] Chung HY, Cesari M, Anton S, Marzetti E, Giovannini S, Seo AY, et al. Molecular inflammation: underpinnings of aging and age-related diseases. Ageing Res Rev 2009;8(1):18-30.

[86] Montezano AC, Touyz RM. Reactive oxygen species, vascular Noxs, and hypertension: focus on translational and clinical research. Antioxid Redox Signal 2014; 20(1):164-82.

[87] Harman D. Aging: a theory based on free radical and radiation chemistry. J Gerontol 1956;11:298-300.

[88] Harman D. Free radical theory of aging: an update: increasing the functional life span. Ann N Y Acad Sci 2006;1067:10-21.

[89] Fan Q, Chen L, Cheng S, Li F, Lau WB, Wang le F, et al. Aging aggravates nitratemediated ROS/RNS changes. Oxid Med Cell Longev 2014;2014:376515.

[90] Tatchum-Talom R1, Martin DS. Tempol improves vascular function in the mesen-teric vascular bed of senescent rats. Can J Physiol Pharmacol 2004;82(3):200-7.

[91] Ungvari Z, Bailey-Downs L, Sosnowska D, Gautam T, Koncz P, Losonczy G. Vascular oxidative stress in aging: a homeostatic failure due to dysregulation of NRF2-mediated antioxidant response. Am J Physiol Heart Circ Physiol 2011;301 :H363-72.

[92] Spencer NF, Poynter ME, Im SY, Daynes RA. Constitutive activation of NF-kappa B in an animal model of aging. Int Immunol 1997;9(10):1581-8.

[93] Krause KH. Aging: a revisited theory based on free radicals generated by NOX family NADPH oxidases. Exp Gerontol 2007;42(4):256-62.

[94] Brandes RP, Weissmann N, Schröder K. Nox family NADPH oxidases: molecular mechanisms of activation. Free Radic Biol Med 2014;76C:208-26.

[95] Cencioni C, Spallotta F, Martelli F, Valente S, Mai A, Zeiher AM, et al. Oxidative stress and epigenetic regulation in ageing and age-related diseases. Int J Mol Sci 2013; 14(9):17643-63.

[96] Wind S, Beuerlein K, Armitage ME, Taye A, Kumar AH, Janowitz D, et al. Oxidative stress and endothelial dysfunction in aortas of aged spontaneously hypertensive rats by NOX1/2 is reversed by NADPH oxidase inhibition. Hypertension 2010; 56(3):490-7.

[97] Touyz RM, Briones AM, Sedeek M, Burger D, Montezano AC. NOX isoforms and reactive oxygen species in vascular health. Mol Interv 2011;11(1):27-35.

[98] Montezano AC, Touyz RM. Molecular mechanisms ofhypertension-reactive oxygen species and antioxidants: a basic science update for the clinician. Can J Cardiol 2012;28(3):288-95.

[99] Montezano AC, Burger D, Ceravolo GS, Yusuf H, Montero M, Touyz RM. Novel Nox homologues in the vasculature: focusing on Nox4 and Nox5. Clin Sci (Lond) 2011; 120(4):131-41.

[100] Eskurza I, Kahn ZD, Seals DR. Xanthine oxidase does not contribute to impaired peripheral conduit artery endothelium-dependent dilatation with ageing. J Physiol 2006;571(Pt 3):661-8.

[101 ] Kokoszka JE, Coskun P, Esposito LA, Wallace DC. Increased mitochondrial oxidative stress in the Sod2 (+/-) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis. Proc Natl Acad Sci U S A 2001 ;98: 2278-83.

[102] Naidoo N, Ferber M, Master M, Zhu Y, Pack AI. Aging impairs the unfolded protein response to sleep deprivation and leads to proapoptotic signaling. J Neurosci 2008; 28:6539-48.

[103] LennaS, Han R, Trojanowska M. Endoplasmic reticulum stress and endothelial dysfunction. IUBMB Life 2014;66(8):530-7.

[104] Panganiban RA,Mungunsukh O,DayRMX-irradiationinduces ERstress, apoptosis, and senescence in pulmonary artery endothelial cells. Int J Radiat Biol 2013;89(8): 656-67.


A. Harvey et al. / Journal of Molecular and Cellular Cardiology xxx (2015) xxx-xxx

912 [105

915 [106

917 [107

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929 [111

933 [112

936 [113

938 [114

940 [115

943 [116

946 [117

949 [118

952 [119

954 [120

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979 [129

Galán M, Kassan M, Kadowitz PJ, Trebak M, Belmadani S, Matrougui K. Mechanism of endoplasmic reticulum stress-induced vascular endothelial dysfunction. Biochim Biophys Acta 2014;1843(6):1063-75.

Najjar SS, Scuteri A, Lakatta EG. Arterial aging: is it an immutable cardiovascular risk factor? Hypertension 2005;46(3):454-62.

Hinojosa-Laborde C, Craig T, Zheng W, Ji H, Haywood JR Sandberg K. Ovariectomy augments hypertension in aging female Dahl salt-sensitive rats. Hypertension 2004;44(4) :405-9.

Savoia C, Burger D, Nishigaki N, Montezano A, Touyz RM. Angiotensin II and the vascular phenotype in hypertension. Expert Rev Mol Med 2011;13:e11. Chugh G, Lokhandwala Mf, Asghar M. Altered functioning of both renal dopamine D1 and angiotensin II type 1 receptors causes hypertension in old rats. Hypertension 2012 May;59(5):1029-36.

Osako MK, Nakagami H, Shimamura M, Koriyama H, Nakagami F, Shimizu H, et al. Cross-talk of receptor activator of nuclear factor-kappaB ligand signaling with renin-angiotensin system in vascular calcification. Arterioscler Thromb Vasc Biol 2013;33(6):1287-96.

Gonzalez GE, Rhaleb NE, D'Ambrosio MA, Nakagawa P, Liu Y, Leung P, et al. Deletion of interleukin-6 prevents cardiac inflammation, fibrosis and dysfunction without affecting blood pressure in angiotensin II-high salt-induced hypertension. J Hypertens 2015;33(1):144-52.

Imanishi M, Tomita S, Ishizawa K, Kihira Y, Ueno M, Izawa-Ishizawa Y, et al. Smooth muscle cell-specific Hif-1alpha deficiency suppresses angiotensin II-induced vascular remodelling in mice. Cardiovasc Res 2014;102(3):460-8. AlGhatrif M, Lakatta EG. The conundrum of arterial stiffness, elevated blood pressure, and aging. Curr Hypertens Rep 2015;17(2):1-9. Montezano AC, Nguyen Dinh Cat A, Rios FJ, Touyz RM. Angiotensin II and vascular injury. Curr Hypertens Rep 2014;16(6):43.

de Cavanagh EMV, Piotrkowski B, Basso N, Stella I, Inserra F, Ferder L, et al. Enalapril and losartan attenuate mitochondrial dysfunction in aged rats. FASEB J 2003;17: 1096-8.

Gohlke P, Linz W, Schölkens BA, Wiemer G, Unger T. Cardiac and vascular effects of long-term losartan treatment in stroke-prone spontaneously hypertensive rats. Hypertension 1996;28(3):397-402.

Brown KA, Didion SP, Andresen JJ, Faraci FM. Effect of aging, MnSOD deficiency, and genetic background on endothelial function: evidence for MnSOD haploinsufficiency. Arterioscler Thromb Vasc Biol 2007;27:1941-6.

Basso N, Paglia N, Stella I, de Cavanagh EM, Ferder L, del Rosario Lores Arnaiz M, et al. Protective effect of the inhibition of the renin-angiotensin system on aging. Regul Pept 2005;128(3):247-52.

Benigni A, Corna D, Zoja C, Sonzogni A, Latini R, Salio M, et al. Disruption of the Ang II type 1 receptor promotes longevity in mice. J Clin Invest 2009;119(3):524-30. Meyer MR, Fredette NC, Barton M, Prossnitz ER. Endothelin-1 but not angiotensin II contributes to functional aging in murine carotid arteries. Life Sci 2014;118(2): 213-8.

Barton M. Aging and endothelin: determinants of disease. Life Sci 2014;118(2): 97-109.

Hannemann A, Wallaschofski H, Lüdemann J, Völzke H, Markus MR, Rettig R et al. Plasma aldosterone levels and aldosterone-to-renin ratios are associated with en-dothelial dysfunction in young to middle-aged subjects. Atherosclerosis 2011; 219(2):875-9.

Paar M, Pavenstädt H, Kusche-Vihrog K, Drüppel V, Oberleithner H, Kliche K. Endothelial sodium channels trigger endothelial salt sensitivity with aging. Hypertension 2014;64(2):391-6.

Brown JM, Underwood PC, Ferri C, Hopkins PN, Williams GH, Adler GK, et al. Aldosterone dysregulation with aging predicts renal vascular function and cardiovascular risk. Hypertension 2014;63(6):1205-11.

Panza JA, Casino PR, Kilcoyne CM, Quyyumi AA Role of endothelium-derived nitric

oxide in the abnormal endothelium-dependent vascular relaxation of patients with

essential hypertension. Circulation 993;87(5):1468-1474.

Sumimoto T, Hamada M, Kawakami H, Suzuki M, Abe M, Matsuoka H, et al. Effects

of glyceryl trinitrate on blood pressure and arterial compliance. Angiology 1993;


Kanno Y, Into T, Lowenstein CJ, Matsushita K. Nitric oxide regulates vascular calcification by interfering with TGF-signalling. Cardiovasc Res 2008;77(1):221-30. Vasa M, Breitschopf K, Zeiher AM, Dimmeler S. Nitric oxide activates telomerase and delays endothelial cell senescence. Circ Res 2000;87(7):540-2. Hayashi T, Matsui-Hirai H, Miyazaki-Akita A, Fukatsu A, Funami J, Ding QF, et al. Endothelial cellular senescence is inhibited by nitric oxide: implications in

atherosclerosis associated with menopause and diabetes. Proc Natl Acad Sci 981 U S A2006;103(45):17018-23. 982

Chunyu Z, Junbao D, Dingfang B, Hui Y, Xiuying T, Chaoshu T. The regulatory effect 983 of hydrogen sulfide on hypoxic pulmonary hypertension in rats. Biochem Biophys 984 Res Commun 2003;302(4):810-6. 985

Ahmad FuD, Sattar MA, Rathore HA, Abdullah MH, Tan S, Abdullah NA, et al. Exog- 986 enous hydrogen sulfide (H2S) reduces blood pressure and prevents the progres- 987 sion of diabetic nephropathy in spontaneously hypertensive rats. Ren Fail 2012; 988 34(2):203-10. 989

Yang G, Zhao K, Ju Y, Mani S, Cao Q, Puukila S, et al. Hydrogen sulfide protects 990 against cellular senescence via S-sulfhydration of Keap1 and activation of Nrf2. 991 Antioxid Redox Signal 2013;18(15):1906-19. 992

Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, et al. H2S as a physiologic vasorelaxant: 993 hypertension in mice with deletion of cystathionine gamma-lyase. Science 2008; 994 322(5901):587-90. 995

Suo R, Zhao Z, Tang Z, Ren Z, Liu X, Liu L, et al. Hydrogen sulfide prevents H2O2- 996 induced senescence in human umbilical vein endothelial cells through SIRT1 acti- 997 vation. Mol Med Rep 2013;7(6):1865-70. 998

Chen Y, Yao W, Geng B, Ding Y, Lu M, Zhao M, et al. Endogenous hydrogen sulfide 999 in patients with COPD. CHEST J 2005;128(5):3205-11. 1000

Zhang Y, Tang ZH, Ren Z, Qu SL, Liu MH, Liu LS, et al. Hydrogen sulfide, the next po- 1001 tent preventive and therapeutic agent in aging and age-associated diseases. Mol 1002 Cell Biol 2013;33(6):1104-13. 1003

Wu S, Pan C, Geng B, Zhao J, Yu F, Pang Y, et al. Hydrogen sulfide ameliorates vas- 1004 cular calcification induced by vitamin D3 plus nicotine in rats. Acta Pharmacol Sin 1005 2006;27(3):299-306. 1006

Zavaczki E, Jeney V, Agarwal A, Zarjou A, Oros M, Katko M, et al. Hydrogen sulfide 1007 inhibits the calcification and osteoblastic differentiation of vascular smooth muscle 1008 cells. Kidney Int 2011;80(7):731-9. 1009

Pan L, Liu X, Gong Q, Wu D, Zhu Y. Hydrogen sulfide attenuated tumor necrosis 1010 factor-a-induced inflammatory signaling and dysfunction in vascular endothelial 1011 cells. PLoS One 2011;6(5):e19766. 1012

Zanardo RC, Brancaleone V, Distrutti E, Fiorucci S, Cirino G, Wallace JL. Hydrogen 1013 sulfide is an endogenous modulator of leukocyte-mediated inflammation. FASEB 1014 J 2006;20(12):2118-20. 1015

Franklin SS, Gustin IV W, Wong ND, Larson MG, Weber MA, Kannel WB, et al. He- 1016 modynamic patterns of age-related changes in blood pressure. The Framingham 1017 Heart Study. Circulation 1997;96(1):308-15.

Miles EA, Rees D, Banerjee T, Cazzola R, Lewis S, Wood R, et al. Age-related increases in circulating inflammatory markers in men are independent of BMI, blood pressure and blood lipid concentrations. Atherosclerosis 2008;196(1):298-305. Blacher J, Guerin AP, Pannier B, Marchais SJ, London GM. Arterial calcifications, arterial stiffness, and cardiovascular risk in end-stage renal disease. Hypertension 2001 ;38(4) :938-42.

AlGhatrif M, Strait JB, Morrell CH, Canepa M, Wright J, Elango P, et al. Longitudinal trajectories of arterial stiffness and the role of blood pressure: the Baltimore Longitudinal Study of Aging. Hypertension 2013;62(5):934-41. Litwin M, Feber J, Niemirska A, Michalkiewicz J. Primary hypertension is a disease of premature vascular aging associated with neuro-immuno-metabolic abnormalities. Pediatr Nephrol Feb. 28 2015 [Epub ahead of print] . Feber J, Ahmed M. Hypertension in children: new trends and challenges. Clin Sci (Lond) 2010;119(4):151-61.

Falkner B. Hypertension in children and adolescents: epidemiology and natural history. Pediatr Nephrol 2010;25(7):1219-24.

Ferreira I, Twisk JW, van Mechelen W, Kemper HC, Seidell JC, Stehouwer CD. Current and adolescent body fatness and fat distribution: relationships with carotid intima-media thickness and large artery stiffness at the age of 36 years. J Hypertens 2004;22(1):145-55.

Wang M, Zhang J, Jiang LQ, Spinetti G, Pintus G, et al. Proinflammatory profile within the grossly normal aged human aortic wall. Hypertension 2007;50:219-27. Wang M, Takagi G, Asai K, Resuello RG, Natividad FF, et al. Aging increases aortic 1041 MMP-2 activity and angiotensin II in nonhuman primates. Hypertension 2003; 1042 41:1308-16.

Wang M, Zhang J, Spinetti G, Jiang LQ, Monticone R, et al. Angiotensin II activates matrix metalloproteinase type II and mimics age-associated carotid arterial remodeling in young rats. Am J Pathol 2005;167:1429-42.

Rimoldi SF, Sartori C, Rexhaj E, Cerny D, Von Arx R, Soria R, et al. Vascular dysfunction in children conceived by assisted reproductive technologies: underlying mechanisms and future implications. Swiss Med Wkly 2014;144:w13973.

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